Establishing Bipotential Human Lung Organoid Culture System and Differentiation to Generate Mature Alveolar and Airway Organoids

A robust in vitro model of the human respiratory epithelium, including the alveolar and the airway epithelium, is essential for understanding the biology and pathology of the human respiratory system. We previously described a protocol to derive human lung organoids from primary lung tissues. We now describe a protocol to induce bidirectional differentiation to generate mature alveolar or airway organoids. The lung organoids are consecutively expanded for over one year with high stability, while the differentiated alveolar and airway organoids morphologically and functionally simulate the human alveolar and airway epithelium to a near-physiological level. Thus, we establish a robust organoid culture system of the entire human respiratory epithelium, the first two-phase bipotential organoid culture system that enables long-term expansion and bidirectional differentiation of respiratory epithelial cells. The long-term expandable lung organoids and differentiated organoids generate a stable and renewable source of respiratory epithelial cells, enabling scientists to reconstruct and expand the human respiratory epithelium in culture dishes. The respiratory organoid system provides a unique and physiologically active in vitro model of the human respiratory epithelium for various applications, including studying respiratory viral infection, disease modeling, drug screening, and pre-clinical testing. Graphical abstract


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Published: Apr 20, 2023 describe a protocol to generate optimized 2D airway organoids from lung organoids, which mimic the airway epithelium more favorably. Altogether, we established a bipotential lung organoid culture system that could enable bidirectional differentiation into alveolar organoids upon distal differentiation or airway organo ids upon proximal differentiation. The lung organoids serve as a stable source for long-term expansion, while differentiated airway and alveolar organoids faithfully phenocopy the human airway and alveolar epithelium, respectively. These organoids are robust and physiologically active tools that are applicable to various experimental manipulations, to explore the biology and pathology of the human lungs.

Derivation of lung organoids
a. Obtain freshly resected lung tissues from patients who underwent surgical operations due to various diseases. Transport the tissue in the cold basal medium (and preferably on ice at 4 °C) and process it as soon as possible.
Note: The lung tissues/biopsies should be resected from the distal lung region, should be composed of normal tissues adjacent to diseased tissues (with both bronchioles and alveoli), and should be ~0.5 cm in diameter. b. Mince the tissue into small pieces (≤1 mm) with a sterile scalpel in a 100 mm cell culture dish. Wash the tissue pieces with 10 mL of cold basal medium and transfer them to a 15 mL centrifuge tube. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant. c. Resuspend the tissue pieces in 8 mL of cold basal medium supplemented with collagenase at a final concentration of 2 mg/mL. Digest the tissue pieces for 30-40 min at 37 °C in a shaking incubator at 120 rpm. d. Shear the digested tissue pieces by pipetting up and down 20 times using a 10 mL serological pipette.
Filter the cell suspension through a 100 μm cell strainer. e. (Optional) Recover remaining tissue pieces from the cell strainer with cold basal medium and transfer them to a 15 mL centrifuge tube for a second round of mechanical shearing by pipetting and then filtering to increase the cell yield. f. Add FBS to the flowthrough with a final concentration of 2% to terminate enzymatic digestion.
Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant. g. (Optional) Resuspend the pellet in 2 mL of erythrocyte lysis buffer and incubate for 5 min at room temperature to remove the red blood cells. h. Wash the cells with 10 mL of cold basal medium. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant. i. Resuspend the pellet in cold matrigel. Add 80-160 μL of matrigel for cells obtained from a lung tissue of size ~0.5 cm in diameter. Dispense 40 μL of the cell suspension to each well of a prewarmed 24well suspension culture plate and incubate for 10-15 min at 37 °C in a standard cell culture CO2 incubator to let the matrigel droplet solidify. j. Add 500 μL of expansion medium to each well and incubate the plate in a standard cell culture CO 2 incubator. Replenish the medium every 2-3 days with caution not to disrupt the matrigel droplets.
Observe the organoids and monitor their growth under a light microscope regularly ( Figure 1).

Notes: 1) Approximately 2.4 × 10 6 cells are required for generating a plate of alveolar organoids in 24-well format. 2) Do not put too many cells in each well for suspension culture because cells may form aggregates if the density is too high, which may compromise alveolar differentiation.
3) Avoid scratching the bottom of the wells during pipetting, which may damage the super low cell attachment surface of the Nunclon Sphera culture plates. j. Incubate the cells in a standard cell culture incubator for 10-14 days for maturation ( Figure 2).
Directly add 100 μL of DD medium to each well every other day without removing the old medium, since evaporation reduces the volume of the medium during incubation. Evenly distribute the cells by gentle shaking before putting the plate back into the incubator. Notes:

Reagent
Final concentration Amount Basal medium n/a Top up to 100 mL Dexamethasone (

Rspondin1 conditioned medium
a. Prepare selection medium (500 mL of DMEM + 60 mL of FBS + 5 mL of penicillin-streptomycin + 1.5 mL of zeocin selection reagent). b. Prepare growing medium (500 mL of DMEM + 60 mL of FBS + 5 mL of penicillin-streptomycin). c. Culture the Rspondin1 expressing 293T cells in a T175 flask in selection medium in a standard cell culture CO2 incubator and wait until they reach confluence. d. Split the cells into six T175 flasks in growing medium and wait until they reach confluence. e. Remove the growing medium and culture the cells in 50 mL basal medium (Recipe 1) for seven days. f. Collect the medium (i.e., the Rspondin1 conditioned medium). Centrifuge at 300 × g for 5 min to pellet the cells/cell debris and filter the medium through a 0.22 μm bottle top vacuum filter. g. Aliquot the medium into small volumes (~50 mL) and store them at -80 °C.

Noggin conditioned medium
a. Prepare selection medium (500 mL of DMEM + 60 mL of FBS + 5 mL penicillin-streptomycin + 5 mL of G418 sulfate). b. Prepare growing medium (500 mL of DMEM + 60 mL of FBS + 5 mL of penicillin-streptomycin). c. Culture the Noggin expressing HEK293 cells in a T175 flask in selection medium in a standard cell culture CO2 incubator and wait until they reach confluence. d. Split the cells into six T175 flasks in growing medium and wait until they reach confluence. e. Remove the growing medium and culture the cells in 50 mL of basal medium (Recipe 1) for seven days. f. Collect the medium (i.e., the Noggin conditioned medium). Centrifuge at 300 × g for 5 min to pellet the cells/cell debris and filter the medium through a 0.22 μm bottle top vacuum filter. g. Aliquot the medium into small volumes (~50 mL) and store them at -80 °C.

WNT3A conditioned medium
a. Prepare selection medium (500 mL of DMEM + 60 mL of FBS + 5 mL penicillin-streptomycin + 625 μL of zeocin). b. Prepare growing medium (500 mL of DMEM + 60 mL of FBS + 5 mL of penicillin-streptomycin). c. Culture the Rspondin1 expressing 293T cells in a T175 flask in selection medium in a standard cell culture CO2 incubator and wait until they reach confluence. d. Split the cells into six T175 flasks in growing medium and wait until they reach confluence. e. Split the cells into thirty 100 mm cell culture dishes in growing medium (20 mL per dish) and culture the cells for seven days. f. Collect the medium (i.e., the WNT3A conditioned medium). Centrifuge at 300 × g for 5 min to pellet the cells/cell debris and filter the medium through a 0.22 μm bottle top vacuum filter. g. Aliquot the medium into small volumes (~50 mL) and store them at -80 °C.